Bladder cancer is very common, costly to manage but extremely poorly studied. The incidence of bladder cancer is disproportionally higher in the Veterans than in the general population because the disease mainly afflicts the aging males with a history of cigarette smoke. The risk of developing bladder cancer is even higher in patients with spinal cord injuries ? a condition very prevalent in the Veterans. There are many challenges in the clinical management of bladder cancer including the unpredictable nature of recurrence and progression of low-grade non-invasive tumors, the aggressiveness of muscle-invasive tumors and their tendency to metastasize despite radical cystectomy, the high frequency of cisplatin resistance and the lack of effective therapy for metastatic disease. Patients with this disease therefore inevitably experience tremendous amount of pain and suffering, high medical expenses and low quality of life. Despite its prevalence, clinical challenges and socioeconomic ramifications, bladder cancer has received a level of attention far below many less common and deadly diseases. Until recently, relatively little research was devoted to bladder cancer and few if any new drugs were approved by the FDA to treat advanced bladder cancer. While the whole genome, exome and transcriptome profiling of human bladder cancer of late has injected new life into the research arena, many of the new findings have not been translated into fundamentally changing the clinical practice. The primary goals of this project are therefore to acquire a deep understanding the genetic and molecular events that play principal roles in bladder cancer formation and progression and then to utilize such information to develop new diagnostic and therapeutic strategies. Toward this overall goal, a range of studies were carried out during the last funding period that focused on isoform 2 of pyruvate kinase (PKM2), a key rate-limiting enzyme controlling aerobic glycolysis or Warburg effect. Major findings include that (i) PKM2 is upregulated in a vast majority of low-grade papillary and high-grade invasive bladder cancer; (ii) RNAi inhibition of PKM2 markedly reduces cell proliferation and increases apoptosis and autophagy; (iii) shikonin binds and inhibits PKM2-mediated tumor cell growth; (iv) shikonin and cisplatin together are much more inhibitory of bladder cancer growth in vitro and in vivo; (v) acquired resistance to cisplatin is strongly associated with PKM2 upregulation; and (vi) constitutive urothelium-specific knockout of PKM2 reduces the formation of low-grade papillary bladder tumors in genetically engineered mice. These and other data demonstrate the critical importance of PKM2 in bladder tumorigenesis and set a stage for work proposed in this renewal application. Three series of studies are designed to broaden and deepen our understanding of the role and mechanisms of action of PKM2 in different pathways and stages of bladder cancer and its therapeutic potentials. The first series will define the tumorigenic stage(s) during which PKM2 is indispensable. Sophisticated mouse genetic engineering approach will be employed to ablate PKM2 in a time-controlled manner before or after the formation of low-grade papillary or high-grade invasive bladder cancer. The second series will examine whether divergent mutations of the RTK-RAS-PI3K, the most prevalently altered pathway in bladder cancer, converge to upregulate PKM2. Overexpression and knockdown of specific mutations in cultured cells will be followed by expression analyses of PKM2 and RNA splicing factors. The third series will determine the functional significance of PKM2 upregulation in cisplatin-resistant bladder cancer cells and whether inhibiting PKM2 by RNAi or shikonin re- sensitize these cells to cisplatin using in vitro and in vivo approaches. Together, these three series of studies should offer novel insights into the role(s) and underlying mechanism(s) of PKM2 overexpression during bladder cancer formation and progression and the therapeutic value of inhibiting PKM2 in reducing cisplatin resistance and in treating various forms of bladder cancer.